Characterization of Estrogen Receptors in the Liver Cytosol of the Rainbow Trout, Salmo Gairdneri

Abstract

Two types of estrogen binding sites were found in the rainbow trout liver cytosol, using the equilibrium binding assay. The higher affinity, lower capacity type I site had a Kd range of 0.53-5.9 nM and a concentration range of 14-95 pmoles/g protein. The lower affinity, higher capacity type II sites had a Kd range of 65-265 nM and a concentration range of 20-180 pmoles/g protein. These estrogen binding sites are both bound by ^3H-moxestrol. The estrogen binding sites in the serum were also examined and two components were found with the higher affinity component having a Kd of 2.63 nM and a concentration of 25 pmoles/g protein while the lower affinity component had a Kd of 79 nM and a concentration of 200 pmoles/g protein. However, these estrogen binding components are unable to bind the synthetic estrogen, DES, and therefore cannot account for either of the binding components found in the liver cytosol which bind DES as readily as 17β-estradiol. The cytosol estrogen binding sites bind non-estrogens in addition to estrogens. Sucrose gradient centrifugation of the cytosol yielded two estrogen binding peaks, one at 4.4S, the other at 3.7S. The heavier peak contained binding sites able to bind progesterone as well. Gel filtration of the cytosol also resulted in two peaks, one at 43,000 daltons, the other at 33,000 daltons. Again the heavier peak could be partially competed out by progesterone. The half life of these binding components was 60 minutes at 37°C, while no decrease in binding was observed after 4 hours at either 0 or 12 0 c. After 17β-estradiol treatment in vivo type I sites (relative to type II sites) were 74% depleted after 8.5 hours and 40% depleted after 24 hours. Finally, o,p'-DDT and p,p'-DDT, components of technical grade preparations of the insecticide DDT were both able to compete for estrogen binding sites in the rainbow trout liver cytosol and therefore may be able to affect the expression of estrogen inducible genes.